Naunyn-Schmiedeberg's
Archivesof Pharmacology
Naunyn-Schmiedeberg's Arch. Pharmacol. 300, 193-194 (1977)
9 by Springer-Verlag 1977
Short Communication
Temperature Dependence of Phalloidin Response in Isolated Rat Hepatocytes M. F R I M M E R and U. RUFEGER Institut ffir Pharmakologie und Toxikologie, Fachbereich Veterin/irmedizin der Justus Liebig-Universit/it Giessen, D-6300 Giessen, Federal Republic of Germany
Summary. Below 21~
isolated hepatocytes are insensitive to phalloidin. Between 21 and 30~ the amount of affected cells increases to nearly maximal values, whereas a further rise of the temperature has little additional effect. These findings suggest that phase transition of membranal lipids might be responsible for the unusual temperature characteristics. The fluidity of lipids seems to control an early step of the phalloidin response.
Key words: Phalloidin poisoning - Isolated hepatocytes - Phase transition - Plasma membranes.
INTRODUCTION Preliminary experiments showed, that the potassium loss from perfused rat livers poisoned with phalloidin was markedly delayed at 20~ (Frimmer, 1972) in comparison with experiments at 37~C. One hour after the addition of 500 lag phalloidin to a perfused rat liver no K+-release was seen at 20 ~ whereas the concentration of potassium in the medium increased by about 2 mVal K+/1 perfusion medium at 40~ after the same period. At 5~ H3-desmethylphalloin was not taken up by perfused livers (Puchinger, 1968). However, for the evaluation of temperature characteristics the perfused liver is an unfit object. It is difficult to compare several individual livers, each tested on a single temperature. Furthermore in absence of red cells the oxygen supply of perfused livers may be sufficient at 25~ whereas hypoxic conditions must be expected at temperatures above 30~ Send offprint requests to M. Frimmer at the above address This work is supported by the Deutsche Forschungsgemeinschaft
In the meantime technics with isolated hepatocytes may satisfy all criteria needed for successful studies of temperature characteristics of the response of hepatocytes to phalloidin.
METHODS Preparation of Hepatocytes. Hepatocytes were prepared according to Berry and Friend (1969) with some modifications (Petzinger and Seeger, 1976). The cell suspension was allowed to equilibrate for 30 rain in slowly shaking Erlenmeyer flasks at 37~ C in presence of 95 % Oz and 5 % CO2 (carbogen). Between 1 0 - 1 5 % of the hepatocytes were stainable with 0.2% trypan blue (non viable cells). Cells were suspended in tyrode solution buffered to pH 7.4 in a final concentration of 2 x 106 cells/ml. Before phalloidin (10 lag/ml) was added, aliquots of 0.2 ml were equilibrated for 5 rain in slowly shaking reaction tubes at different temperatures in presence of carbogen. In each series 37~ was the control temperature. Quantitative Analysis of Phalloidin Poisoning in Isolated Hepatocytes. About 100 cells were counted after an incubation period of 20, 30 and 60 rain in a Bfirker-Ttirk Chamber. The quantity of phalloidin response was expressed as percentage of viable cells which formed protrusions after treatment with phalloidin. Only cells not stainable by trypan blue were counted, because only viable cells react with phalloidin.
RESULTS The best criterium of phalloidin poisoning in isolated liver cells is the development of typical protrusions on their surface (Weiss et al., 1973). The ratio affected cells/total viable cells is a good criterium for the quantity of response, as shown in earlier papers (Frimmer et al., 1974, 1975; Petzinger, 1975 ; Petzinger et al., 1975). Figure I and Table 1 demonstrate the dependence on temperature of the phalloidin response under standard conditions. Within a period of 20 min no marked effects of the toxin were seen up to 21 ~C. Between 21 and 30 ~C the ratio of affected cells jumps
194 100 -
Naunyn-Schmiedeberg's Arch. Pharmacol. 300 (1977)
"/~CeLlswith protrusbns
fO
9080-
/
70605040302010-
#. . /
0
~i 2'0 21
2'3
2'5
30
3'7~
Fig. I. Dependence on temperature of the phalloidin response. Hepatocytes from a single preparation were incubated with phal-
loidin at different temperatures. After 20 min the ratio of affected cells/total viable cells was determined Table 1. Temperature dependence of phalloidin response in isolated rat hepatocytes After After 60 min 20 min Formation of protrusions, measured at 15~ at 18~ at 20~
0% 0% 0%
0% (n = 3) 6.0+_ 5.6% (n=3) 62.9 _+18.2% (n = 8)
Lowest temperature starting phalloidin response, measured after 20 min
21.0_+0.8~
Average of temperature leading to 50 % affected cells
23.8_+ 1.2~ (n = 14)
(n = 7)
to 80 % o f the m a x i m u m (Fig. 1). T h a t means that the response o f hepatocytes to phalloidin does n o t follow an usual continous temperature coefficient. I n the range between 1 5 - 2 1 ~ the response to phalloidin was n o t fully suppressed but m a r k e d l y delayed (Table 1). H o w e v e r below 15~ no response to phalloidin could be seen even after incubation periods o f 60 rain. I f cells were exposed to phalloidin at 10~ during 10 min and then incubated at 3 7 ~ after washing o u t the n o n b o u n d toxin, no response was seen after 20 a n d 60 rain. This is in agreement with the results o f Puchinger (1968). A p p a r e n t l y phalloidin does not bind to the acceptor below 21~ DISCUSSION A t present no other a p p r o a c h is probable than to assume that lipid structures participate in the response o f liver cells to phalloidin. H o w e v e r o u r results give no informations, in which step o f the response lipids m a y be involved.
F r o m experiments with phospholipase A we k n o w that any degradation o f phosphatides on the surface o f hepatocytes reduces phalloidin sensitivity in a reversible manner. (Petzinger et al., 1977, in press). On the other h a n d no evidence for phalloidin binding to phosphatides could be detected. Therefore we expect that a protein, responsible for the recognition o f phalloidin, m a y be inserted in a specific lipid area. The latter might be a specific d o m a i n e with characteristic behavior o f phase transition. F u r t h e r evidence for the protein character o f the phalloidin acceptor were detected by experiments with trypsin ( F r i m m e r et al., 1977, in press) and with several protein specific reagents at low concentration (unpublished). Earlier experiments (Puchinger, 1968) on perfused rat livers suggest that the resistance o f liver cells at low temperatures against phalloidin is caused by a block of uptake, not by inhibition o f subsequent reactions. REFERENCES Berry, M. N., Friend, D. S.: High-yield preparation of isolated rat liver parenchymal cells. J. Cell Biol. 43, 506-520 (1969) Frimmer, M.: Temperature dependence of potassium depletion in the phalloidin poisoned perfused rat liver. Naunyn-Schmiedeberg's Arch. Pharmacol. 272, 354-357 (1972) Frimmer, M. : The role of plasma membranes in hepatotoxic effects. Pharmacology Meeting, Hannover 1976, NaunynSchmiedeberg's Arch. Pharmacol. 297, S 15- S 19 (1977) Frimmer, M., Kroker, R., Porstend6rfer, J.: The mode of action of phalloidin: Demonstration of rapid deformation of isolated hepatocytes by scanning electron microscopy. NaunynSchmiedeberg's Arch. Pharmacol. 284, 395-398 (1974) Frimmer, M., Petzinger, E., Homann, J. : Phalloidin-Antagonisten. 4. Mitteilung: Thiocts/iure, SH-Verbindungen, Rifampicin, Choleretika, Dexamethason, ()stradiol, unspezifische Hemmstoffe und unwirksame Verbindungen. Arzneim.-Forsch. (Drug Res.) 25, 1881-1884 (1975) Frimmer, M., Petzinger, E., Rufeger, U., Veil, L. B.: Trypsin protection of hepatocytes against phalloidin. Naunyn-Schmiedeberg's Arch. Pharmacol. 300, 163-171 (1977) Petzinger, E.: Die Vergiftung isolierter Rattenhepatozyten mit Phalloidin und deren Beeinflussung durch Trypsin und Phalloidinantagonisten. Vet.-Med. Diss., Giegen 1975 Petzinger, E., Seeger, R. : Scanning electron microscopic studies on the cytolytic effect of phallolysin on isolated rat hepatocytes and AS-30 D hepatoma cells. Naunyn-Schmiedeberg's Arch. Pharmacol. 295, 211 --213 (1976) Petzinger, E., Homann, J., Frimmer, M. : Phalloidin-Antagonisten. 2. Mitteilung: Protektive Wirkung von Disilybin bei der Vergiftung isolierter Hepatozyten mit Phalloidin. Arzneim.-Forsch. (Drug Res.) 25, 571- 576 (1975) Petzinger, E., Rufeger, U., Frimmer, M.: Transient desensibilization of isolated hepatocytes against phalloidin by treatment with phospholipase A. Naunyn-Schmiedeberg's Arch. Pharmacol. (in press, 1977) Puchinger, H. : Zum Wirknngsmechanismus yon Phalloidin und Antamanid. Inaug.-Diss., Frankfurt/M. 1968 (Naturwiss. Fak.) Weiss, E., Sterz, I., Frimmer, M., Kroker, R. : Electron microscopy of isolated rat hepatocytes before and after treatment with phalloidin. Beitr. Path. 150, 345-356 (1973) Received June 20/Accepted September 7, 1977
Archivesof Pharmacology
Naunyn-Schmiedeberg's Arch. Pharmacol. 300, 193-194 (1977)
9 by Springer-Verlag 1977
Short Communication
Temperature Dependence of Phalloidin Response in Isolated Rat Hepatocytes M. F R I M M E R and U. RUFEGER Institut ffir Pharmakologie und Toxikologie, Fachbereich Veterin/irmedizin der Justus Liebig-Universit/it Giessen, D-6300 Giessen, Federal Republic of Germany
Summary. Below 21~
isolated hepatocytes are insensitive to phalloidin. Between 21 and 30~ the amount of affected cells increases to nearly maximal values, whereas a further rise of the temperature has little additional effect. These findings suggest that phase transition of membranal lipids might be responsible for the unusual temperature characteristics. The fluidity of lipids seems to control an early step of the phalloidin response.
Key words: Phalloidin poisoning - Isolated hepatocytes - Phase transition - Plasma membranes.
INTRODUCTION Preliminary experiments showed, that the potassium loss from perfused rat livers poisoned with phalloidin was markedly delayed at 20~ (Frimmer, 1972) in comparison with experiments at 37~C. One hour after the addition of 500 lag phalloidin to a perfused rat liver no K+-release was seen at 20 ~ whereas the concentration of potassium in the medium increased by about 2 mVal K+/1 perfusion medium at 40~ after the same period. At 5~ H3-desmethylphalloin was not taken up by perfused livers (Puchinger, 1968). However, for the evaluation of temperature characteristics the perfused liver is an unfit object. It is difficult to compare several individual livers, each tested on a single temperature. Furthermore in absence of red cells the oxygen supply of perfused livers may be sufficient at 25~ whereas hypoxic conditions must be expected at temperatures above 30~ Send offprint requests to M. Frimmer at the above address This work is supported by the Deutsche Forschungsgemeinschaft
In the meantime technics with isolated hepatocytes may satisfy all criteria needed for successful studies of temperature characteristics of the response of hepatocytes to phalloidin.
METHODS Preparation of Hepatocytes. Hepatocytes were prepared according to Berry and Friend (1969) with some modifications (Petzinger and Seeger, 1976). The cell suspension was allowed to equilibrate for 30 rain in slowly shaking Erlenmeyer flasks at 37~ C in presence of 95 % Oz and 5 % CO2 (carbogen). Between 1 0 - 1 5 % of the hepatocytes were stainable with 0.2% trypan blue (non viable cells). Cells were suspended in tyrode solution buffered to pH 7.4 in a final concentration of 2 x 106 cells/ml. Before phalloidin (10 lag/ml) was added, aliquots of 0.2 ml were equilibrated for 5 rain in slowly shaking reaction tubes at different temperatures in presence of carbogen. In each series 37~ was the control temperature. Quantitative Analysis of Phalloidin Poisoning in Isolated Hepatocytes. About 100 cells were counted after an incubation period of 20, 30 and 60 rain in a Bfirker-Ttirk Chamber. The quantity of phalloidin response was expressed as percentage of viable cells which formed protrusions after treatment with phalloidin. Only cells not stainable by trypan blue were counted, because only viable cells react with phalloidin.
RESULTS The best criterium of phalloidin poisoning in isolated liver cells is the development of typical protrusions on their surface (Weiss et al., 1973). The ratio affected cells/total viable cells is a good criterium for the quantity of response, as shown in earlier papers (Frimmer et al., 1974, 1975; Petzinger, 1975 ; Petzinger et al., 1975). Figure I and Table 1 demonstrate the dependence on temperature of the phalloidin response under standard conditions. Within a period of 20 min no marked effects of the toxin were seen up to 21 ~C. Between 21 and 30 ~C the ratio of affected cells jumps
194 100 -
Naunyn-Schmiedeberg's Arch. Pharmacol. 300 (1977)
"/~CeLlswith protrusbns
fO
9080-
/
70605040302010-
#. . /
0
~i 2'0 21
2'3
2'5
30
3'7~
Fig. I. Dependence on temperature of the phalloidin response. Hepatocytes from a single preparation were incubated with phal-
loidin at different temperatures. After 20 min the ratio of affected cells/total viable cells was determined Table 1. Temperature dependence of phalloidin response in isolated rat hepatocytes After After 60 min 20 min Formation of protrusions, measured at 15~ at 18~ at 20~
0% 0% 0%
0% (n = 3) 6.0+_ 5.6% (n=3) 62.9 _+18.2% (n = 8)
Lowest temperature starting phalloidin response, measured after 20 min
21.0_+0.8~
Average of temperature leading to 50 % affected cells
23.8_+ 1.2~ (n = 14)
(n = 7)
to 80 % o f the m a x i m u m (Fig. 1). T h a t means that the response o f hepatocytes to phalloidin does n o t follow an usual continous temperature coefficient. I n the range between 1 5 - 2 1 ~ the response to phalloidin was n o t fully suppressed but m a r k e d l y delayed (Table 1). H o w e v e r below 15~ no response to phalloidin could be seen even after incubation periods o f 60 rain. I f cells were exposed to phalloidin at 10~ during 10 min and then incubated at 3 7 ~ after washing o u t the n o n b o u n d toxin, no response was seen after 20 a n d 60 rain. This is in agreement with the results o f Puchinger (1968). A p p a r e n t l y phalloidin does not bind to the acceptor below 21~ DISCUSSION A t present no other a p p r o a c h is probable than to assume that lipid structures participate in the response o f liver cells to phalloidin. H o w e v e r o u r results give no informations, in which step o f the response lipids m a y be involved.
F r o m experiments with phospholipase A we k n o w that any degradation o f phosphatides on the surface o f hepatocytes reduces phalloidin sensitivity in a reversible manner. (Petzinger et al., 1977, in press). On the other h a n d no evidence for phalloidin binding to phosphatides could be detected. Therefore we expect that a protein, responsible for the recognition o f phalloidin, m a y be inserted in a specific lipid area. The latter might be a specific d o m a i n e with characteristic behavior o f phase transition. F u r t h e r evidence for the protein character o f the phalloidin acceptor were detected by experiments with trypsin ( F r i m m e r et al., 1977, in press) and with several protein specific reagents at low concentration (unpublished). Earlier experiments (Puchinger, 1968) on perfused rat livers suggest that the resistance o f liver cells at low temperatures against phalloidin is caused by a block of uptake, not by inhibition o f subsequent reactions. REFERENCES Berry, M. N., Friend, D. S.: High-yield preparation of isolated rat liver parenchymal cells. J. Cell Biol. 43, 506-520 (1969) Frimmer, M.: Temperature dependence of potassium depletion in the phalloidin poisoned perfused rat liver. Naunyn-Schmiedeberg's Arch. Pharmacol. 272, 354-357 (1972) Frimmer, M. : The role of plasma membranes in hepatotoxic effects. Pharmacology Meeting, Hannover 1976, NaunynSchmiedeberg's Arch. Pharmacol. 297, S 15- S 19 (1977) Frimmer, M., Kroker, R., Porstend6rfer, J.: The mode of action of phalloidin: Demonstration of rapid deformation of isolated hepatocytes by scanning electron microscopy. NaunynSchmiedeberg's Arch. Pharmacol. 284, 395-398 (1974) Frimmer, M., Petzinger, E., Homann, J. : Phalloidin-Antagonisten. 4. Mitteilung: Thiocts/iure, SH-Verbindungen, Rifampicin, Choleretika, Dexamethason, ()stradiol, unspezifische Hemmstoffe und unwirksame Verbindungen. Arzneim.-Forsch. (Drug Res.) 25, 1881-1884 (1975) Frimmer, M., Petzinger, E., Rufeger, U., Veil, L. B.: Trypsin protection of hepatocytes against phalloidin. Naunyn-Schmiedeberg's Arch. Pharmacol. 300, 163-171 (1977) Petzinger, E.: Die Vergiftung isolierter Rattenhepatozyten mit Phalloidin und deren Beeinflussung durch Trypsin und Phalloidinantagonisten. Vet.-Med. Diss., Giegen 1975 Petzinger, E., Seeger, R. : Scanning electron microscopic studies on the cytolytic effect of phallolysin on isolated rat hepatocytes and AS-30 D hepatoma cells. Naunyn-Schmiedeberg's Arch. Pharmacol. 295, 211 --213 (1976) Petzinger, E., Homann, J., Frimmer, M. : Phalloidin-Antagonisten. 2. Mitteilung: Protektive Wirkung von Disilybin bei der Vergiftung isolierter Hepatozyten mit Phalloidin. Arzneim.-Forsch. (Drug Res.) 25, 571- 576 (1975) Petzinger, E., Rufeger, U., Frimmer, M.: Transient desensibilization of isolated hepatocytes against phalloidin by treatment with phospholipase A. Naunyn-Schmiedeberg's Arch. Pharmacol. (in press, 1977) Puchinger, H. : Zum Wirknngsmechanismus yon Phalloidin und Antamanid. Inaug.-Diss., Frankfurt/M. 1968 (Naturwiss. Fak.) Weiss, E., Sterz, I., Frimmer, M., Kroker, R. : Electron microscopy of isolated rat hepatocytes before and after treatment with phalloidin. Beitr. Path. 150, 345-356 (1973) Received June 20/Accepted September 7, 1977
